Progestins (P) are major mitogens in the adult human breast and contribute significantly to breast cancer risk. Since P are widely used in oral contraceptives and in postmenopausal hormone replacement therapy it is critically important that their mechanims of action be understood. It is the goal of the proposed research to delineate the mechanisms of P-dependent proliferation in the normal mammary gland. In the current grant period, using the mouse mammary gland model, we have developed a minimally-supplemented, serum-free collagen gel primary culture system in which mammary epithelial organoids, made up from both luminal epithelial and myoepithelial cells, proliferate and undergo alveolar morphogenesis in response to P. In addition to P, these responses require mammary stroma derived-hepatocyte growth factor (HGF). Treatment with HGF alone induces proliferation and tubulo-ductal morphogenesis, whereas P alone does not induce proliferation but induces lumen formation that is associated with increased apoptosis. HGF+P increase proliferation above HGF alone and cause a change from ductal to alveolar morphology. This is a novel in vitro demonstration of a mitogenic and morphological response to P that recapitulates the proliferative and alveologenesis response to P in vivo. In addition a pro-apoptotic response to P in normal mammary cells has been identified. In this proposal we will determine whether and how P acts in the cytoplasm, possibly through an SH3 binding domain on the progesterone receptor (PR), to affect HGF/Met-activated pathways associated with mammary-specific tublogenesis (FAK, SHIP-1, PI3-K) and proliferation (ERK, Src/Myc) to inhibit tubulogenesis, promote proliferation and cause alveologenesis. We will also determine the effect of PR action in the nucleus, via its transcriptional activation function, on the expression of P-responsive target genes relevant to alveologenesis (Wnt-4) and proliferation (Met, cyclin D1, Myc). In addition will also identify the specific cells types (luminal epithelial, myoepithelial cells) in which these effects of P occur. Using cultures derived from PRA null or PRB null mice we will also determine the role of the two PR isoforms (A and B) in proliferation and alveologenesis. The long-term goal of this proposal is the delineation of the mechanisms of P-dependent growth regulation in the adult normal mammary gland that may lead to novel strategies for the prevention and treatment of breast cancer.